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CMV infection is a significant complication after solid organ transplantation. We used single cell TCR αβ sequencing to determine how memory inflation impacts clonality and diversity of the CMV-responsive CD8 and CD4 T cell repertoire in the first year after transplantation in human subjects. We observed CD8 T cell inflation but no changes in clonal diversity, indicating homeostatic stability in clones. In contrast, the CD4 repertoire was diverse and stable over time, with no evidence of CMV-responsive CD4 T cell expansion. We identified shared CDR3 TCR motifs among patients but no public CMV-specific TCRs. Temporal changes in clonality in response to transplantation and in the absence of detectable viral reactivation suggest changes in the repertoire immediately after transplantation followed by an expansion with stable clonal competition that may mediate protection.

CMV is a major infectious complication following solid organ transplantation. Reactivation of CMV leads to memory inflation, a process in which CD8 T cells expand over time. Memory inflation is associated with specific changes in T cell function, including increased oligoclonality, decreased cytokine production, and terminal differentiation. To address whether memory inflation during the first year after transplantation in human subjects alters T cell differentiation and function, we employed single-cell–matched TCRαβ and targeted gene expression sequencing. Expanded T cell clones exhibited a terminally differentiated, immunosenescent, and polyfunctional phenotype whereas rare clones were less differentiated. Clonal expansion occurring between pre- and 3 mo posttransplant was accompanied by enhancement of polyfunctionality. In contrast, polyfunctionality and differentiation state were largely maintained between 3 and 12 mo posttransplant. Highly expanded clones had a higher degree of polyfunctionality than rare clones. Thus, CMV-responsive CD8 T cells differentiated during the pre- to posttransplant period then maintained their differentiation state and functional capacity despite posttransplant clonal expansion.

Regulatory B cells (Bregs) can suppress immune responses through the secretion of IL-10 and other anti-inflammatory cytokines. This immunomodulatory capacity holds therapeutic potential, yet a definitional immunophenotype for enumeration and prospective isolation of B cells capable of IL-10 production remains elusive. We therefore applied mass cytometry to simultaneously quantify cytokine production and immunophenotype in human peripheral B cells across a range of stimulatory conditions and timepoints. While multiple B cell subsets produced IL-10, no phenotype uniquely identified IL-10+ B cells and a significant portion of IL-10+ B cells co-expressed the proinflammatory cytokines IL-6 and TNFα. Despite this heterogeneity, we found operationally tolerant liver transplant recipients had a unique enrichment of IL-10+, but not TNFα+ or IL-6+, B cells as compared to transplant recipients receiving immunosuppression. Thus, human IL-10-producing B cells constitute an induced, transient state arising from a diversity of B cell subsets that may contribute to maintenance of immune homeostasis.

Stem cell-based approaches have the potential to address the organ shortage in transplantation. Whereas both embryonic stem cells and induced pluripotent stem cells have been utilized as cellular sources for differentiation and lineage specification, their relative ability to be recognized by immune effector cells is unclear. We determined the expression of immune recognition molecules on hepatocyte-like cells (HLC) generated from murine embryonic stem cells and induced pluripotent stem cells, compared to adult hepatocytes, and we evaluated the impact on recognition by natural killer (NK) cells. We report that HLC lack MHC class I expression, and that embryonic stem cell-derived HLC have higher expression of the NK cell activating ligands Rae1, H60, and Mult1 than induced pluripotent stem cell-derived HLC and adult hepatocytes. Moreover, the lack of MHC class I renders embryonic stem cell-derived HLC, and induced pluripotent stem cell-derived HLC, susceptible to killing by syngeneic and allogeneic NK cells. Both embryonic stem cell-derived HLC, and induced pluripotent stem cell-derived HLC, are killed by NK cells at higher levels than adult hepatocytes. Finally, we demonstrate that the NK cell activation receptor, NKG2D, plays a key role in NK cell cytotoxicity of embryonic stem cell-derived HLC, but not induced pluripotent stem cell-derived HLC.

Epstein-Barr virus (EBV) is a ubiquitous, yet perplexing, gammaherpesvirus. Primary infection with EBV can have widely diverse outcomes ranging from asymptomatic infection to self-resolving infectious mononucleosis, to the development of EBV-associated malignancies including Burkitt lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma, and B cell lymphoma in transplant recipients and AIDS patients. In this chapter we will discuss the biology of EBV and its life cycle, viral genetics, and oncogenic mechanisms – and how these factors impact on the pathogenesis of PTLD.

Alloimmune responses in acute rejection are complex, involving multiple interacting cell types and pathways. Deep profiling of these cell types has been limited by technology that lack the capacity to resolve this high dimensionality. Single-cell mass cytometry was used to characterize the alloimmune response in early acute rejection, measuring 37 parameters simultaneously, across multiple time-points in two models; a well-established murine cardiac and a novel murine vascularized composite allotransplant (VCA). Unsupervised hierarchical clustering was used to group related cell types defined by combinatorial expression of surface and intracellular proteins, along with markers of effector function and activation. This expression profile was mapped to visualize structural changes in antigen composition across cell types, revealing unique phenotypic signatures in alloimmune T cells, NK cells, and myeloid subsets that were conserved and that firmly distinguished rejecting from non-rejecting grafts.

BackgroundLiver failure of unknown etiology (LFUE) has a transplant-free survival rate MethodsWe identified all children who underwent liver transplant for LFUE at a single quaternary children’s hospital; 51/65 cases could be age matched with controls (children who underwent liver transplant for metabolic liver disease). Quantitative polymerase chain reaction for HHV-6 was performed on DNA from formalin-fixed paraffin-embedded liver explant tissue.ResultsHHV-6 was detected in 34/51 cases (66.7%) and 19/51 controls (37.3%) (P = .005). Average HHV-6 viral load was 213207 copies/106 cells in positive cases (range: 7293–1102030) and 38115 copies/106 cells in positive controls (range: 1382–122375) (P = .0008). HHV-6 was present significantly more often in cases compared to controls in patients younger than 6 years. In particular, in patients younger than 3 years, HHV-6 was present in 13/27 cases (48.1%) and 2/27 controls (7.4%) (P = .0009).ConclusionsHHV-6 was detected in liver explants significantly more often and in higher quantities in children transplanted for LFUE compared to controls, suggesting HHV-6 should be evaluated in young children who present with LFUE.

Epstein-Barr Virus (EBV) is associated with potentially fatal lymphoproliferations such as post-transplant lymphoproliferative disorder (PTLD), a serious complication of transplantation. The viral mechanisms underlying the development and maintenance of EBV+ B cell lymphomas remain elusive but represent attractive therapeutic targets. EBV modulates the expression of host microRNAs (miRs), non-coding RNAs that regulate gene expression, to promote survival of EBV+ B cell lymphomas. Here, we examined how the primary oncogene of EBV, latent membrane protein 1 (LMP1), regulates host miRs using an established model of inducible LMP1 signaling. LMP1 derived from the B95.8 lab strain or PTLD induced expression of the oncogene miR-155. However, PTLD variant LMP1 lost the ability to upregulate the tumor suppressor miR-193. Small molecule inhibitors (SMI) of p38 MAPK, NF-κB, and PI3K p110α inhibited upregulation of miR-155 by B95.8 LMP1; no individual SMI significantly reduced upregulation of miR-155 by PTLD variant LMP1. miR-155 was significantly elevated in EBV+ B cell lymphoma cell lines and associated exosomes and inversely correlated with expression of the miR-155 target FOXO3a in cell lines. Finally, LMP1 reduced expression of FOXO3a, which was rescued by a PI3K p110α SMI. Our data indicate that tumor variant LMP1 differentially regulates host B cell miR expression, suggesting viral genotype as an important consideration for the treatment of EBV+ B cell lymphomas. Notably, we demonstrate a novel mechanism in which LMP1 supports the regulation of miR-155 and its target FOXO3a in B cells through activation of PI3K p110α. This mechanism expands on the previously established mechanisms by which LMP1 regulates miR-155 and FOXO3a and may represent both rational therapeutic targets and biomarkers for EBV+ B cell lymphomas.

Epstein-Barr Virus (EBV) is a ubiquitous virus linked to a variety of lymphoid and epithelial malignancies. In solid organ and hematopoietic stem cell transplant recipients, EBV is causally associated with posttransplant lymphoproliferative disorder (PTLD), a group of heterogeneous lymphoid diseases. EBV(+) B cell lymphomas that develop in the context of PTLD are generally attributed to the immunosuppression required to promote graft survival, but little is known regarding the role of EBV genome diversity in the development of malignancy. We deep-sequenced the EBV genome from the peripheral blood of 18 solid organ transplant recipients, including 6 PTLD patients. Sequences from 6 EBV(+) spontaneous lymphoblastoid B cell lines (SLCL) were similarly analyzed. The EBV genome from PTLD patients had a significantly greater number of variations than EBV from transplant recipients without PTLD. Importantly, there were 15 nonsynonymous variations, including 8 in the latent cycle gene EBNA3C that were associated with the development of PTLD. One of the nonsynonymous variations in EBNA3C is located within a previously defined T cell epitope. These findings suggest that variations in the EBV genome can contribute to the pathogenesis of PTLD.

Although the liver is less immunogenic than other solid organs, most liver transplant recipients receive lifelong immunosuppression. In both experimental models and clinical transplantation, total lymphoid irradiation (TLI) has been shown to induce allograft tolerance. Our goal was to identify the microRNAs (miRNAs) expressed in tolerant liver allograft recipients in an experimental model of TLI-induced tolerance.To identify the miRNAs associated with TLI-induced tolerance, we examined syngeneic recipients (Lewis→Lewis) and allogeneic recipients (Dark Agouti→Lewis) of orthotropic liver transplants that received posttransplant TLI, allogeneic recipients that were not treated posttransplantation and experienced acute rejection, and native Dark Agouti livers. Quantitative-polymerase chain reaction miRNA array cards were used to profile liver grafts.We identified 12 miRNAs that were specifically and significantly increased during acute rejection. In early tolerance, 33 miRNAs were altered compared with syngeneic livers, with 80% of the miRNAs increased. In established tolerance, 42 miRNAs were altered. In addition, miR-142-5p and miR-181a demonstrated increased expression in tolerant livers (both early and established tolerance) as compared with syngeneic livers. A principal component analysis of all miRNAs assayed demonstrated a profile in established tolerance that was closely related to that seen in syngeneic livers.The miRNA profile of established tolerant allografts is very similar to syngeneic grafts, suggesting tolerance may be a return to an immunological state of quiescence.

T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual1–4. Here we define the minimal requirements for TCR antigen specificity, through an analysis of TCR sequences using a panel of peptide and major histocompatibility complex (pMHC)-tetramer-sorted cells and structural data. From this analysis we developed an algorithm that we term GLIPH (grouping of lymphocyte interactions by paratope hotspots) to cluster TCRs with a high probability of sharing specificity owing to both conserved motifs and global similarity of complementarity-determining region 3 (CDR3) sequences. We show that GLIPH can reliably group TCRs of common specificity from different donors, and that conserved CDR3 motifs help to define the TCR clusters that are often contact points with the antigenic peptides. As an independent validation, we analysed 5,711 TCRβ chain sequences from reactive CD4 T cells from 22 individuals with latent Mycobacterium tuberculosis infection. We found 141 TCR specificity groups, including 16 distinct groups containing TCRs from multiple individuals. These TCR groups typically shared HLA alleles, allowing prediction of the likely HLA restriction, and a large number of M. tuberculosis T cell epitopes enabled us to identify pMHC ligands for all five of the groups tested. Mutagenesis and de novo TCR design confirmed that the GLIPH-identified motifs were critical and sufficient for shared-antigen recognition. Thus the GLIPH algorithm can analyse large numbers of TCR sequences and define TCR specificity groups shared by TCRs and individuals, which should greatly accelerate the analysis of T cell responses and expedite the identification of specific ligands.

Natural killer (NK) cells are effector cells of the innate immune system that can lyse target cells without prior sensitization and have an important role in host defense to pathogens and transformed cells. A balance between negative and positive signals transmitted via germ line-encoded inhibitory and activating receptors controls the function of NK cells. Although the concept of "missing-self" would suggest that NK cells could target foreign allografts, the prevailing dogma has been that NK cells are not active participants in the mechanisms that culminate in the rejection of solid organ allografts. Recent studies, however, challenge this conclusion and instead implicate NK cells in contributing to both graft rejection and tolerance to an allograft. In this review, we highlight recent studies with the goal of understanding the complex NK cell interactions that impact alloimmunity.

MicroRNAs (miRNAs) are important regulators in many biologic processes and have been implicated in the control of genes relevant to acute rejection and liver functions. Here we review the miRNAs specifically expressed in allografts during acute rejection and discuss potential roles for these miRNAs in liver dysfunction. We focus on miRNAs dysregulated both in the liver and in peripheral blood mononuclear cells and include a discussion of the potential for these miRNAs as non-invasive biomarkers to reflect liver status posttransplant.

Cell replacement therapy holds promise for a number of untreatable neurological or psychiatric diseases but the immunogenicity of cellular grafts remains controversial. Emerging stem cell and reprogramming technologies can be used to generate autologous grafts that minimize immunological concerns but autologous grafts may carry an underlying genetic vulnerability that reduces graft efficacy or survival. Healthy allogeneic grafts are an attractive and commercially scalable alternative if immunological variables can be controlled. Stem cells and immature neural progenitor cells (NPC) do not express major histocompatibility complex (MHC) antigens and can evade adaptive immune surveillance. Nevertheless, in an experimental murine model, allogeneic NPCs do not survive and differentiate as well as syngeneic grafts, even when traditional immunosuppressive treatments are used. In this study, we show that natural killer (NK) cells recognize the lack of self-MHC antigens on NPCs and pose a barrier to NPC transplantation. NK cells readily target both syngeneic and allogeneic NPC, and killing is modulated primarily by NK-inhibiting “self” class I MHC and NK-activating NKG2D-ligand expression. The absence of NKG2D signaling in NK cells significantly improves NPC-derived neuron survival and differentiation. These data illustrate the importance of innate immune mechanisms in graft outcome and the potential value of identifying and targeting NK cell-activating ligands that may be expressed by stem cell derived grafts.

Single-cell flow cytometric techniques have been indispensable to improving our understanding of the phenotype and function of immune cell subsets that are important in both rejection and tolerance post-transplant. Mass cytometry, or Cytometry by Time-of-Flight (CyTOF), is a single cell–based platform that utilizes antibodies conjugated to rare heavy metal ions for analysis of cellular proteins by a time-of-flight mass spectrometer. This new technology allows for the evaluation of over 40 simultaneous cellular parameters in a single sample because the limitation of spectral overlap, seen in conventional flow cytometry, is eliminated. In this review we discuss the current state of mass cytometry, describe the advantages and disadvantages over multi-parameter flow cytometry, introduce novel methods of high-dimensional data analysis and visualization, and review some recent studies using mass cytometry to profile the immune system of healthy people, and transplant recipients.

MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate the posttranscriptional expression of target genes and are important regulators in immune responses. Previous studies demonstrated that the miRNA, miR-182 was significantly increased during allograft rejection. Further, the transcription factor Forkhead box (FOX) protein 1, (FOXO1) was shown to be a target of miR-182. The aim of this study is to further examine the role of miR-182 in alloimmune responses.Transplantation of BALB/c cardiac allografts was performed in C57BL/6, miR-182, B6.129S-H2 (MHC II and CD4 T cell-deficient) and B6.129S2-Tap1 (MHC I and CD8 T cell-deficient) mice, with or without CTLA-4Ig administration. T cell phenotype, FOXO1 protein levels and graft infiltrating lymphocytes were determined in C57BL/6 or miR-182 mice by flow cytometric analysis, Western blot, and immunohistochemistry, respectively.We now show that T cells, mainly CD4 are the main cellular source of miR-182 during allograft rejection. In the absence of miR-182, CTLA-4Ig treatment significantly increased allograft survival (31.5 days C57BL/6 vs 60 days miR-182; P0.01). Further, CTLA4-Ig treatment inhibits miR-182 expression, increases FOXO1 levels, and reduces the percentage of CD4CD44 T cells after transplantation. Fewer T cells infiltrate the cardiac allografts, and memory T cells are significantly decreased in allograft recipients deficient in miR-182 with CTLA4-Ig treatment (P0.01).Our findings suggest that miR-182 contributes to the T-cell responses to alloantigen especially under costimulation blockade. Therapeutics that target specific miRNAs may prove beneficial in transplantation.

Epstein-Barr virus (EBV) is a human -herpesvirus that establishes latency and lifelong infection in host B cells while achieving a balance with the host immune response. When the immune system is perturbed through immunosuppression or immunodeficiency, however, these latently-infected B cells can give rise to aggressive B cell lymphomas. Natural killer (NK) cells are regarded as critical in the early immune response to viral infection, but their role in controlling expansion of infected B cells is not understood. Here we report that NK cells from healthy human donors display increased killing of autologous B lymphoblastoid cell lines (LCL) harboring latent EBV compared to primary B cells. Co-culture of NK cells with autologous EBV+ LCL identifies an NK cell population that produces IFN-gamma and mobilizes the cytotoxic granule protein CD107a. Multi-parameter flow cytometry and Boolean analysis reveals that these functional cells are enriched for expression of the NK cell receptor NKG2A. Further, NKG2A+ NK cells more efficiently lyse autologous LCL than do NKG2A- NK cells. More specifically, NKG2A+2B4+CD16-CD57-NKG2C-NKG2D+ cells constitute the predominant NK cell population that responds to latently-infected autologous EBV+ B cells. Thus, a subset of NK cells is enhanced for the ability to recognize and eliminate autologous, EBV-infected transformed cells, laying the groundwork for harnessing this subset for therapeutic use in EBV+ malignancies.

Post-transplant lymphoproliferative disorders (PTLDs) are a group of conditions that involve uncontrolled proliferation of lymphoid cells as a consequence of extrinsic immunosuppression after organ or haematopoietic stem cell transplant. PTLDs show some similarities to classic lymphomas in the non-immunosuppressed general population. The oncogenic Epstein-Barr virus (EBV) is a key pathogenic driver in many early-onset cases, through multiple mechanisms. The incidence of PTLD varies with the type of transplant; a clear distinction should therefore be made between the conditions after solid organ transplant and after haematopoietic stem cell transplant. Recipient EBV seronegativity and the intensity of immunosuppression are among key risk factors. Symptoms and signs depend on the localization of the lymphoid masses. Diagnosis requires histopathology, although imaging techniques can provide additional supportive evidence. Pre-emptive intervention based on monitoring EBV levels in blood has emerged as the preferred strategy for PTLD prevention. Treatment of established disease includes reduction of immunosuppression and/or administration of rituximab (a B cell-specific antibody against CD20), chemotherapy and EBV-specific cytotoxic T cells. Despite these strategies, the mortality and morbidity remains considerable. Patient outcome is influenced by the severity of presentation, treatment-related complications and risk of allograft loss. New innovative treatment options hold promise for changing the outlook in the future.

MicrorRNA are small noncoding RNA molecules that regulate the posttranscriptional expression of target genes. In addition to being involved in many biologic processes, microRNAs are important regulators in innate and adaptive immune responses. Distinct sets of expressed microRNAs are found in different cell types and tissues and aberrant expression of microRNAs is associated with many disease states. MicroRNA expression was examined in a model of heterotopic heart transplantation by microarray analyses and a unique profile was detected in rejecting allogeneic transplants (BALB/c → C57BL/6) as compared to syngeneic transplants (C57BL/6 → C57BL/6). The microRNA miR-182 was significantly increased in rejecting cardiac allografts and in mononuclear cells that infiltrate the grafts. Forkhead box (FOX) proteins are a family of important transcription factors and FOXO1 is a target of miR-182. As miR-182 increases after transplant, there is a concomitant posttranscriptional decrease in FOXO1 expression in heart allografts that is localized to both the cardiomyocytes and CD3(+) T cells. The microRNA miR-182 is significantly increased in both peripheral blood mononuclear cells and plasma during graft rejection suggesting potential as a biomarker of graft status. Our results identify microRNAs that may regulate alloimmune responses and graft outcomes.

PurposeIn transplant recipients, Epstein-Barr Virus (EBV) is associated with the development of B cell lymphomas in post-transplant lymphoproliferative disorder (PTLD). We utilized next-generation sequencing to characterize the diversity of the EBV genome in transplant recipients with low EBV viral

Epstein Barr virus (EBV) is associated with B-cell lymphomas in posttransplant lymphoproliferative disease (PTLD). Latent membrane protein 1 (LMP1), the major oncogenic protein of EBV, promotes tumorigenesis through activation of NF-kappaB, Erk, p38, JNK and Akt. The Jak/STAT signal transduction pathway is also constitutively active in PTLD-associated EBV(+) B-cell lymphomas. Here we determine the mechanism of Jak/STAT activation in EBV(+) B-cell lymphomas and the role of LMP1 in this process. Immunoprecipitation studies revealed no direct interaction of LMP1 and JAK3, but known associations between JAK3 and common gamma chain, and between LMP1 and TRAF3, were readily detected in EBV(+) B cell lines from patients with PTLD. An inducible LMP1 molecule expressed in EBV(-) BL41 Burkitt's cells demonstrated STAT activation only after prolonged LMP1 signaling. While LMP1 induced IFN-gamma production in BL41 cells, IFN-gamma receptor blockade and IFN-gamma neutralization prior to LMP1 activation markedly decreased STAT1 activation and expression of LMP1-driven IFN-gamma inducible genes. Understanding the mechanisms by which EBV induces cellular signal transduction pathways may facilitate development of new treatments for PTLD.

To summarize recent advances that contribute to our understanding of the pathobiology of Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD), the host immune response to virally infected B cells, and the molecular basis for the effects of mammalian target of rapamycin inhibitors on EBV+ B-cell lymphomas.Cytogenetic and genomic analyses support the concept that the underlying biology of EBV-associated PTLD is complex. Transplant recipients can generate and maintain significant populations of EBV-specific CD8+ memory T cells but the function of these cells may be impaired. EBV invokes multiple strategies to subvert and evade the host immune response. The phosphoinositide-3 kinase/Akt/mammalian target of rapamycin signal transduction pathway is a nexus for growth and survival signals in PTLD-associated EBV+ B-cell lymphomas.Multiple factors influence the development of EBV-associated PTLD including the host immune response to EBV, virally induced effects on the infected cell and the host immune system, and the type and intensity of immunosuppression.

Background. Posttransplant lymphoproliferative disease (PTLD) is a serious complication of solid organ and bone marrow transplantation and is closely associated with Epstein-Barr virus (EBV) infection. We have previously shown that rapamycin (RAPA) directly inhibits the in vitro and in vivo proliferation of EBV-infected B lymphoblastoid cell lines (SLCL), derived from patients with PTLD, by arresting cells in the G1 phase of the cell cycle. The aim of this study is to elucidate the mechanism by which RAPA causes cell cycle arrest in EBV + B cells. Methods. SLCL were cultured without or with RAPA (10 ng/ml) and G1-associated cell cycle proteins were analyzed by immunoblot and densitometric analysis. CDK complexes were immunoprecipitated and incubated with retinoblastoma protein (Rb) substrate. Kinase activity of the complex was determined by Western blot with anti-phospho-Rb antibodies. Results. We show that RAPA decreased both Cyclin D2 and Cyclin D3 protein levels. Furthermore, RAPA decreased the protein levels of cyclin dependent kinase 4 (CDK4) and increased the expression of the CDK inhibitor p27. In contrast, expression of the CDK inhibitor p21 was markedly inhibited by RAPA in the SLCL. Finally, in vitro kinase assays revealed that downstream hyperphosphorylation of Rb by CDK complexes was also decreased by RAPA. Conclusion. The results presented here elucidate key targets of RAPA-induced cell cycle arrest, provide insight into the growth pathways or EBV + B-cell lymphomas, and demonstrate the potential for RAPA as a therapeutic option in the treatment of PTLD and other EBV + lymphomas.

Epstein-Barr virus (EBV) infection is linked to approximately 90% of B-cell lymphomas associated with posttransplant lymphoproliferative disease (PTLD), a serious complication for immunosuppressed transplant recipients. In this paper, we review the myriad ways by which EBV can inadvertently drive the genesis and persistence of B-cell lymphomas, particularly when the antiviral immune response is compromised. Probing the basic mechanisms by which EBV infection proceeds and contributes to malignancy in such cases will hopefully improve our understanding and treatment of PTLD and other EBV-associated malignancies.

Natural killer (NK) cells play an important role in first-line defense against tumor and virus-infected cells. The activity of NK cells is tightly regulated by a repertoire of cell surface expressed inhibitory and activating receptors. NKp46 is a major NK cell-activating receptor that is involved in the elimination of target cells. NK cells form different types of synapses that result in distinct functional outcomes: cytotoxic, inhibitory, and regulatory. Recent studies revealed that complex integration of NK receptor signaling controls cytoskeletal rearrangement and other immune synapse-related events. However, the distinct nature by which NKp46 participates in NK immunological synapse formation and function remains unknown. In this study, we determined that NKp46 forms microclusters structures at the immune synapse between NK cells and target cells. Over-expression of human NKp46 is correlated with increased accumulation of F-actin mesh at the immune synapse. Concordantly, knock-down of NKp46 in primary human NK cells decreased recruitment of F-actin to the synapse. Live cell imaging experiments showed a linear correlation between NKp46 expression and lytic granules polarization to the immune synapse. Taken together, our data suggest that NKp46 signaling directly regulates the NK lytic immune synapse from early formation to late function.

The relationship between EBV infection and sensitivity to death receptor (DR)-induced apoptosis is poorly understood. Using EBV− and EBV+ BJAB cells, we provide the first evidence that EBV can protect latently infected B cell lymphomas from apoptosis triggered through Fas or TRAIL receptors. Caspase 8 activation was impaired and cellular FLIP recruitment was enriched in death-inducing signaling complexes formed in EBV-infected BJAB cells relative to parent BJAB cells. Furthermore, latent membrane protein 1 expression alone could reduce caspase activation and confer partial resistance to DR apoptosis in BJAB cells. This protective effect was dependent on C-terminal activating region 2-driven NF-κB activation, which in turn up-regulated cellular FLIP expression in latent membrane protein 1+ BJAB cells. Thus, the ability of latent EBV to block DR apoptosis may help to ensure the survival of host cells during B cell differentiation, and contribute to the development of B cell lymphomas, especially in immunocompromised individuals.

NKp30 is a stimulatory receptor on human NK cells implicated in tumor immunity, and is capable of promoting or terminating dendritic cell maturation. To gain a better understanding of NKp30 biology, we have investigated the expression and function of rat NKp30 (rNKp30). We generated stable transfectants of rNKp30 in RNK16 cells, a rat NK lymphoma line, and used a novel panel of mAb against rNKp30 to study this receptor. Using agonistic rNKp30 mAb, we demonstrated that rNKp30 mediates robust IFN-gamma production and cytolytic responses from rNKp30-transfected RNK16 cells. We determined by flow cytometry that rNKp30 is expressed by a subset of primary NK cells isolated from the blood and spleen, and to a lesser extent also on liver NK cells. Stimulation of rNKp30 on primary NK cells led to IFN-gamma production. Liver NK cells expressed low levels of NKp30 and had reduced rNKp30-mediated IFN-gamma responses. During an alloimmune response in vivo, the proportion of the rNKp30(+) NK cell subset in the peripheral blood significantly increased, suggesting that rNKp30 may play an important role during alloactivation. Thus, our data demonstrate that NKp30 is indeed expressed in rodents and is a functional stimulatory receptor in a subset of rat NK cells.

CD30 is an inducible member of the TNFR superfamily that is expressed on activated T and B cells and some lymphoid malignancies. We have previously shown that human CD30+ T cells elicited with allogeneic APC are a major source of IFN-γ and IL-5 production. In the present study we have used alloantigen, as well as anti-CD3 plus anti-CD28 mAb stimulation, to further characterize human CD30+ T cells with respect to function and the expression of other activation-dependent cell surface molecules, including the related TNFR family members OX-40 and 4-1BB (CD137). Our results indicate that human CD30+ T cells are a subset of activated T cells that also express CD25 and CD45RO. Moreover, we observed that allogeneic APC consistently induced a greater proportion of CD30+ cells within the activated T cell population than did stimulation with plate-bound anti-CD3 plus anti-CD28 mAb or stimulation with soluble anti-CD3 plus anti-CD28 and autologous APC. The enhanced induction of CD30 expression by alloantigen was not common to other inducible TNFR family members because anti-CD3 plus anti-CD28 mAbs were far more effective in inducing expression of 4-1BB and OX-40. Furthermore, CD30 expression marked the predominant proliferating T cell population induced by alloantigen as determined by CFSE staining and flow cytometry. These results indicate that CD30, but not 4-1BB or OX-40, is preferentially induced by alloantigen, suggesting that CD30 may be important in human alloimmune responses.

Background Posttransplant lymphoproliferative disease (PTLD) is a major complication after bone marrow and solid organ transplantation. The disease encompasses a spectrum of abnormal, Epstein-Barr virus (EBV)-associated B-cell proliferations. We have previously shown that EBV-infected, spontaneous lymphoblastoid cell lines (SLCL) derived from PTLD patients require autocrine interleukin (IL)-10 to proliferate. To determine if cytokine signal transduction is involved in the autonomous growth of the SLCL, the activation states of the Jak/STAT signaling pathway proteins were analyzed in three different SLCL, termed JB7, MF4, and VB4. Methods The tyrosine phosphorylation (P-tyr) states of the Janus kinases (Jaks) and signal transducers and activators of transcription (STAT) proteins were examined by immunoprecipitation and immunoblot. Activated STAT dimer formation was determined by electromobility shift assays. Results All three SLCL, but not the Daudi Burkitt's lymphoma B-cell line, expressed the four known Jak kinases constitutively tyrosine phosphorylated, with particularly high levels of P-tyr Jak1 in the JB7 line. STAT1 and STAT3, but not STAT2 or STAT5, are also constitutively activated in all SLCL. The ability of the activated STAT proteins to form DNA-binding dimers was confirmed by electromobility shift assay. The SLCL, but not the Daudi line, express activated STAT complexes composed of STAT1 and STAT3. Another EBV-infected B-cell line, isolated from a lymph node biopsy after kidney transplantation, is phenotypically similar to the other SLCL in both surface antigen and activated STAT1 and STAT3 expression. Conclusion These data support the presence of a constitutively active autocrine signaling pathway consistent with IL-10 in the SLCL.

Human B cells are the primary targets of Epstein–Barr virus (EBV) infection. In most cases, EBV infection is asymptomatic because of a highly effective host immune response, but some individuals develop self-limiting infectious mononucleosis, while others develop EBV-associated lymphoid or epithelial malignancies. The viral and immune factors that determine the outcome of infection are not understood. The EBV life cycle includes a lytic phase, culminating in the production of new viral particles, and a latent phase, during which the virus remains largely silent for the lifetime of the host in memory B cells. Thus, in healthy individuals, there is a tightly orchestrated interplay between EBV and the host that allows the virus to persist. To promote viral persistence, EBV has evolved a variety of strategies to modulate the host immune response including inhibition of immune cell function, blunting of apoptotic pathways, and interfering with antigen processing and presentation pathways. In this article, we focus on mechanisms by which dysregulation of the host B cell and immune modulation by the virus can contribute to development of EBV+ B cell lymphomas.

Background/Aims: Autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are two autoimmune diseases with unknown etiologies that primarily target the liver. In both diseases, liver lesions are accompanied by large infiltrates of mononuclear cells. The purpose of this study was to determine if either the Fas-mediated or the granule-exocytosis pathways contribute to apoptosis in these diseases. Methods: To determine the involvement of apoptosis in tissue injury we examined liver tissue for DNA fragmentation and morphological characteristics of apoptosis. The major cytotoxic pathways of activated lymphocytes were compared by quantitating the levels of transcripts for FasL and granzyme B, and expression was confirmed by immunoprecipitation of these molecules. Results: In both diseases, apoptosis was observed. However, the main cell types undergoing apoptosis were hepatocytes in AIH, and biliary epithelial cells in PBC. In AIH the levels of FasL and granzyme B mRNA were increased over the levels detected in normal liver, while in PBC only the levels of granzyme B were elevated. Additionally, in AIH, the ratio of FasL transcripts to granzyme B transcripts was elevated, reflecting a possible increase in the relative contribution of FasL to the progression of the disease. Immunoprecipitation studies further support an increase in FasL protein expression in AIH. Conclusions: These data suggest that both FasL and granzyme B contribute to the apoptosis observed in AIH and PBC. However, FasL appears to play a more prominent role in the induction of hepatocyte apoptosis and tissue destruction in AIH.

Background. The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We previously demonstrated that CD8 + T cells are not necessary for allograft rejection or for the induction of apoptosis in rat small intestinal transplantation. In this study, we examined the mechanisms of apoptosis and rejection after liver transplantation in the absence of CD8 + T cells. Methods. Either Lewis or dark agouti rat liver grafts were transplanted into Lewis recipients to create syngeneic and allogeneic combinations. CD8 + T cells were depleted in an additional allogeneic group by treatment with OX-8 mAb on day -1 and day 1 after liver transplant. Results. Apoptosis and rejection were observed in both the CD8 + T cell-depleted allogeneic and allogeneic grafts by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and radiolabeled-annexin V in vivo imaging. Granzyme B and FasL were expressed in all allogeneic transplants, including those depleted of CD8 + T cells, indicating that a mononuclear cell other than a CD8 + T cell can be the source of these molecules during allograft rejection. Activation of the caspase cascade was detected in all rejecting allografts. Caspases 3, 8, and 9 were activated at similar significantly elevated levels in both allogeneic and CD8 + T cell-depleted liver grafts. Conclusion. These data indicate that in the absence of CD8 + T cells an alternative pathway, associated with granzyme B and FasL expression and activation of the caspase cascade, can mediate apoptosis and graft rejection.

The Fas/Fas ligand (FasL) pathway has been shown to be important in T lymphocyte-mediated cell death and is a key peripheral immunoregulatory mechanism that limits expansion of antigen-activated lymphocytes. The expression of Fas by commonly transplanted organs such as the heart, lung, kidney, and liver suggests that these tissues may be targets of FasL-expressing allospecific cytotoxic T lymphocytes. In this review the current literature examining the Fas/FasL system as a potential cellular effector pathway in tissue injury is discussed. In addition to a deleterious role in destruction of graft tissue, Fas/FasL interactions may have a beneficial role in transplantation. Recent studies suggest that modulation of FasL in target tissue leads to deletion, via apoptosis, of graft infiltrating lymphoid cells. However, an equally compelling series of reports indicate that overexpression of FasL can lead to a heightened immune response. These data are reviewed in the context of strategies to achieve long term allograft survival.

Epstein-Barr virus (EBV) is a γ-herpesvirus that is linked to the development of posttransplant lymphoproliferative disorder (PTLD) in solid organ recipients. We previously demonstrated that EBV(+) B cell lymphoma cell lines isolated from patients with PTLD produce human IL-10 as an autocrine growth factor. However, little is known regarding IL-10 regulation in B cells. Here we show that EBV infection markedly alters the expression of host B cell microRNA, a class of small noncoding RNA that is an important regulator of transcriptional and posttranscriptional gene expression. Gene arrays reveal unique microRNA profiles in EBV(+) B cell lymphoma lines from patients with PTLD, compared to normal B cells or in vitro generated EBV(+) lymphoblastoid cell lines. We show that microRNA-194 expression is uniquely suppressed in EBV(+) B cell lines from PTLD patients and that the 3'untranslated region of IL-10 is targeted by microRNA-194. Overexpression of microRNA-194 attenuates IL-10 production and increases apoptosis of EBV(+) B cell lymphoma lines. Together, these data indicate that EBV co-opts the host B cell microRNA network and specifically suppresses microRNA-194 to override control of IL-10 expression. Thus, modulation of microRNA-194 may constitute a novel approach to inhibiting proliferation of EBV(+) B cell lymphomas in PTLD.

Background. CD30 is a member of the tumor necrosis factor/nerve growth factor receptor family and has been proposed as a marker of specific cytokine-producing subsets in humans. Previous studies have examined the expression of CD30 on established T helper type 1 and T helper type 2 cell clones and the function of CD30 + cells after mitogenic stimulation. In this study, we examined the development and function of CD30 + T cells generated in response to alloantigen. Methods. Primary one-way mixed lymphocyte reactions were established, and the expression of CD30 on T lymphocytes was determined by immunofluorescence and flow cytometry. Fluorescence-activated cell sorting was utilized to define the cytokine profile of alloactivated CD30 + cells after restimulation with anti-CD3 monoclonal antibodies or alloantigen. The effect of cyclosporine on the development of CD30 + cells, and on cytokines produced by CD30 + T lymphocytes, in response to alloantigen was determined. Results. CD30 + T lymphocytes could be detected on day 2 of mixed lymphocyte reactions and continued to increase in number and proportion through day 6. Both CD4 and CD8 T cells expressed CD30 after primary alloantigenic stimulation. CD30 + T cells are a subset of alloactivated T cells and are the major source of interferon-γ and interleukin-5 produced in response to alloantigen. Cyclosporine partially, but not completely, inhibits the development of CD30 + cells, and has a greater effect on interferon-γ production than on interleukin-5 production. Conclusions. CD30 + T lymphocytes may constitute an important immunoregulatory subset in human allograft rejection.

Background. Transplant patients receiving immunosuppressive drugs are at increased risk for Epstein-Barr virus (EBV)-associated disorders including posttransplant lymphoproliferative disorder. The function of T lymphocytes, which are critical to preventing the expansion of EBV-infected B cells, is inhibited by immunosuppressive drugs. The purpose of this study was to determine whether immunosuppressive drugs have direct effects on EBV-infected B cells. Methods. The growth and proliferation of EBV-infected spontaneous lymphoblastoid cell lines (SLCLs), cultured in the presence or absence of cyclosporine (CsA) and tacrolimus (TAC), were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and[3H]thymidine incorporation assays. The effect of CsA and TAC on the viability of SLCLs was determined by cell counts with trypan blue. Apoptosis of SLCLs was induced with an anti-Fas agonist monoclonal antibody in the presence or absence of CsA and TAC and measured by flow cytometry after terminal deoxynucleotidyl transferase end-labeling and propidium iodide staining. Results. CsA and TAC, but not sirolimus, increased the growth of SLCLs. The increased growth in the presence of CsA and TAC was attributable to enhanced cell viability and not increased cell division of SLCLs. In addition, CsA and TAC inhibited Fas-mediated apoptosis of SLCLs. Conclusions. CsA and TAC enhance the survival of EBV-transformed B-cell lines. CsA and TAC promote or augment SLCL growth through protection from cell death but do not affect cell division. The inhibition of cell death by CsA and TAC may contribute to the expansion of EBV-infected cells in immunosuppressed individuals.

Background. Fas (Apo-1/CD95), a member of the tumor necrosis factor receptor family, can mediate apoptosis when engaged by its ligand or by anti-Fas anti-body. Fas is expressed by cells of the immune system and by some nonlymphoid tissues. Numerous studies have suggested that the Fas pathway may play a role in the rejection of allografts. Functional, soluble forms of the Fas receptor are produced by activated peripheral blood mononuclear cells and some transformed cell lines. The purpose of this study was to determine if soluble variants of Fas are produced in the liver and to determine if blockade of the Fas pathway, by liver-derived soluble Fas, inhibits Fas-mediated apoptosis. Methods. Liver and purified hepatocyte specimens were analyzed for Fas transcripts by reverse transcriptase-polymerase chain reaction with primers that span the transmembrane region of the molecule. Bile and cell lysates were analyzed for soluble Fas by specific enzyme-linked immunosorbent assay. Lysates were prepared from normal liver and hepatocytes and utilized to block Fas-mediated apoptosis of Jurkat cells as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and flow cytometry. Results. A variant form of Fas is abundantly expressed in normal liver and purified hepatocytes. This variant form of Fas is expressed in all normal liver specimens but only in half of the liver specimens obtained during allograft rejection. The levels of soluble Fas diminish in patients undergoing liver allograft rejection in contrast to patients with stable grafts. Importantly, a soluble form of Fas is produced in the liver by hepatocytes and can specifically inhibit Fas-mediated apoptosis. Conclusion. These data raise the possibility that soluble Fas, produced by hepatocytes, may influence the immune response by blocking Fas-mediated apoptosis and, thus, may have a role in liver transplantation.

The delineation of T helper cell subsets into T helper type 1 (Th1) and T helper type 2 (Th2) populations based on the production of specific cytokines has been useful in understanding the regulation and progression of immune-based pathologies. In order to test the relevance of this concept to human solid organ transplantation, in situ and system Th1 and Th2 cytokine profiles were characterized in liver allograft recipients. Bile and serum samples obtained posttransplant were analyzed for the cytokines IL-2, IFN-gamma, IL-4, and IL-10. Significant elevations of IL-4 and IL-10 were measured at the site of graft rejection. In contrast, IL-2, IFN-gamma, and IL-4 were markedly elevated in the circulation during rejection. Analyses of sequential bile samples revealed that Th1 and Th2 cytokines showed similar kinetics of production in response to alloantigen. Taken together, these results indicate that acute rejection of human allografts can proceed [correction of procede] in the presence of minimal levels of the Th1 cytokines IL-2 and IFN-gamma and high levels of IL-4 and IL-10.

Antimitochondrial antibodies (AMA) which recognize the E2 component of the pyruvate dehydrogenase complex are found in virtually all patients with the autoimmune liver disease primary biliary cirrhosis (PBC). The factors that contribute to elevated AMA and the relationship of the autoantibodies to disease pathogenesis have not been elucidated. Since cytokines are important regulators of antibody production and isotype switching, the association of specific cytokines to antibody production was examined in patients with PBC. Elevations in IL-2, IFN-gamma, IL-4, IL-5, and IL-10 were detected in serum from patients with PBC. However, only IFN-gamma (6012 +/- 1128 pg/ml vs 147 +/- 89 pg/ml, P0.0001) and IL-5 (382 +/- 103 pg/ml vs 29 +/- 12 pg/ml, P0.001) were significantly elevated compared to normal controls. Moreover, there was a positive correlation in the levels of IFN-gamma, and to a lesser extent IL-5, with the levels of soluble CD30 (sCD30) in the circulation. The elevated levels of sCD30 detected in patients with PBC (194 +/- 29 U/ml vs 39 +/- 9 U/ml in normal controls) suggest that CD30+ cells may produce cytokines, which contribute to the immune abnormalities in patients with PBC.

T lymphocytes and immunoregulatory cytokines may be important in the host response to hepatitis C virus (HCV) infection. T-helper type 1 (Th1) cytokines (interleukin [IL]-2, interferon gamma [IFN-gamma]) are required for host antiviral immune responses, including cytotoxic T-cell generation and natural killer cell activation, while T-helper type 2 (Th2) cytokines (IL-4,IL-10) can inhibit the development of these effector mechanisms. In this study, the serum levels of Th1 and Th2 cytokines in patients (n = 23) infected with HCV were measured and compared with biochemical (alanine transaminase [ALT]) and viral (HCV RNA) indicators of infection. Serial cytokine levels were measured in a subset of 11 patients at 1 and 12 weeks during and at 1 week after interferon alfa (IFN-α) therapy (n = 33 samples). Levels of circulating IL-2, IL-4, IL-10, and IFN-γ were significantly elevated in HCV patients versus normal controls (128 vs. 25 pg/mL, 3,045 vs. 29 pg/mL, 2,949 vs. 18 pg/mL, and 307 vs. 24 pg/mL respectively; P < .01). Treatment with IFN-α decreased the levels of IL-4 (321 ± 224 pg/mL), and IL-10 (1,011 ± 344 pg/mL), which paralleled a decrease in HCV RNA (114 ± 27 vs. 25 ± 20 Eq/ml X 105, pre- vs. post-IFN-α [12 weeks];P

To determine the immune processes involved in chronic liver allograft rejection (CR) we examined in situ cytokine production in tissue from 15 patients with both clinical and histopathological diagnoses of CR. Total RNA was isolated from liver samples, reverse-transcribed and analyzed by RT-PCR for the production of proinflammatory cytokines and immunoregulatory mediators. Transcripts for the Thl-like cytokines IL-2 and IFN-γ were detected in 53.3% and 46.7% of CR grafts, while they were detected in only 16% and 0% of stable grafts, respectively. The cytotoxic T cell mediator granzyme B was expressed in the majority of liver grafts undergoing CR, but was expressed only in a minority of stable grafts (80% vs. 16%, P

Cytotoxic T lymphocytes (CTL) constitute a major component of the alloreactive response following organ transplantation. The molecular mechanisms of CTL killing remain to be determined but multiple candidate molecules involved in CTL-mediated cytotoxicity have been identified. Granzyme B, a serine protease, participates in perforin-dependent pathways of cytotoxicity and is necessary for induction of DNA fragmentation in target cells. In this study the expression of granzyme B in liver biopsies obtained from liver allograft recipients was determined by semiquantitative reverse transcriptase polymerase chain reaction. Biopsies were classified into four groups—no evidence of rejection, preservation injury, acute rejection, or resolving rejection—according to histopathological criteria. There was a significantly higher frequency of transcripts for granzyme B in the acute rejection group (82.8%) compared to the no rejection (20.0%), resolving rejection (12.5%) and preservation injury (0%) groups. Analysis of granzyme B gene expression in sequential samples from individual patients prior to, and after, treatment for rejection revealed an inverse correlation between granzyme B mRNA and response to treatment. These findings indicate that the cytopathic mediator granzyme B may participate in CTL-mediated cytotoxicity during liver allograft rejection.

Pediatric allograft recipients in particular are at increased risk for Epstein-Barr virus (EBV)-associated disorders. Early identification and diagnosis of EBV-associated disorders is critical, since disease progression can often be halted by reduction of immunosuppression. In this study we examined viral and immunologic parameters of EBV infection in the circulation of pediatric liver recipients to identify factors associated with disease. Peripheral blood DNA from pediatric liver recipients was analyzed by PCR for the EBV genes coding for the nuclear antigen 1 (EBNA-1) and the viral capsid antigen gp220. Sequences for these viral genes could be readily detected in the circulation of 36.5% of patients. Moreover, identification of the EBV genome was associated with symptomatic infection, suggesting that circulating EBV may be a useful marker of disease. Since EBV-infected B cells release the low-affinity IgE receptor (sCD23), we measured sCD23 in the circulation of pediatric liver recipients and found it to be elevated in patients with detectable virus or symptoms of infection. However, sCD23 was also elevated in cases where no EBV was detectable, suggesting that factors other than viral infection could stimulate release of sCD23. To further characterize the immune response to EBV infection, the peripheral levels of IL-4, IL-5, IL-10, and IFN-gamma were determined in pediatric liver recipients. Each of these cytokines was elevated in patients with symptoms or circulating virus compared with stable, age-matched liver recipients. IL-4, in particular, was significantly increased, indicating an important role for this cytokine in EBV infection. Together, these findings suggest that (1) monitoring circulating levels of EBV may be useful in patients at high risk and (2) cytokines that promote B cell growth and differentiation contribute to EBV-associated disorders.

During allograft rejection, adhesion molecules play an integral role in infiltration, activation, and binding of effector cells to target tissue. Some adhesion molecules, including ICAM-1 and VCAM-1, exist in soluble, circulating forms that retain ligand-binding activity. In the present study the levels of circulating ICAM-1 (cICAM-1) and VCAM-1 (cVCAM-1) were compared in the serum and bile of pediatric liver recipients. The cICAM-1 was significantly elevated in the serum during allograft rejection and infection relative to periods when no rejection was apparent. Biliary cICAM-1, however, was specifically elevated during rejection and not during infection or when no rejection was apparent. The cVCAM-1 levels were elevated in the serum during rejection compared with levels when no rejection was evident. In contrast, cVCAM-1 was not detected in the bile. Serum levels of both cICAM-1 and cVCAM-1 decreased rapidly following successful treatment for rejection, whereas elevated levels persisted, or increased, in ongoing rejection. The differential patterns of the circulating forms of ICAM-1 and cVCAM-1 were consistent with the membrane expression of these molecules during graft rejection. ICAM-1 expression was extensive on bile duct epithelium, endothelium, hepatocytes, and infiltrating leukocytes during rejection, while VCAM-1 was restricted to endothelium. These findings indicate that the release of circulating adhesion molecules is a prominent feature of liver allograft rejection. Measurement of these markers may be useful in distinguishing rejection from infection and in determining the efficacy of treatment for rejection

Primary biliary cirrhosis (PBC) is an autoimmune disease of the liver with unknown etiology. Autoreactive T lymphocytes that infiltrate the liver may play a major role in the bile duct damage that accompanies the disease. We hypothesized that cytokines produced by T lymphocytes and other cells are central to the disease process. Therefore, we used reverse transcription-polymerase chain reaction (PCR) and Southern hybridization to identify cytokine message directly from liver tissue of 11 patients with PBC and 5 patients with autoimmune hepatitis (AI-CAH). Messenger RNA (mRNA) for interleukin (IL)-2, IL-5, IL-6, interferon gamma (IFN-gamma), and transforming growth factor beta (TGF-beta) were detected in the majority of the specimens from patients with PBC. The presence of IL-5 was associated with PBC (P < .001, PBC vs. AI-CAH). Because IL-5 is a potent eosinophil differentiation factor, we looked for evidence of activated eosinophils within the infiltrate. We observed the deposition of the primary cytotoxic granule protein of eosinophils, major basic protein (MBP), within the portal region of livers from patients with PBC. Moreover, we detected message for a cytotoxic T-lymphocyte (CTL) granzyme in 87.5% of these livers indicating that mature CTL are present. Thus, we present evidence for two effector pathways that may contribute to the tissue damage observed in PBC and have identified message for cytokines that may regulate these pathways.

The B lymphotrophic γ-herpesvirus EBV is associated with a variety of lymphoid- and epithelial-derived malignancies, including B cell lymphomas in immunocompromised and immunosuppressed individuals. The primary oncogene of EBV, latent membrane protein 1 (LMP1), activates the PI3K/Akt pathway to induce the autocrine growth factor, IL-10, in EBV-infected B cells, but the mechanisms underlying PI3K activation remain incompletely understood. Using small molecule inhibition and siRNA strategies in human B cell lines expressing a chimeric, signaling-inducible LMP1 protein, nerve growth factor receptor (NGFR)-LMP1, we show that NGFR-LMP1 utilizes Syk to activate PI3K/Akt signaling and induce IL-10 production. NGFR-LMP1 signaling induces phosphorylation of BLNK, a marker of Syk activation. Whereas Src kinases are often required for Syk activation, we show here that PI3K/Akt activation and autocrine IL-10 production by NGFR-LMP1 involves the Src family kinase Fyn. Finally, we demonstrate that NGFR-LMP1 induces phosphorylation of c-Cbl in a Syk- and Fyn-dependent fashion. Our results indicate that the EBV protein LMP1, which lacks the canonical ITAM required for Syk activation, can nevertheless activate Syk, and the Src kinase Fyn, resulting in downstream c-Cbl and PI3K/Akt activation. Fyn, Syk, and PI3K/Akt antagonists thus may present potential new therapeutic strategies that target the oncogene LMP1 for treatment of EBV+ B cell lymphomas.

B cell lymphoma survival requires tonic or ligand-independent signals through activation of Syk by the B cell receptor. The Epstein-Barr virus (EBV) protein latent membrane 2a (LMP2a), a mimic of the B cell receptor, provides constitutive survival signals for latently infected cells through Syk activation; however, the precise downstream mechanisms coordinating this survival response in EBV+ B cell lymphomas remain to be elucidated. Herein, we assess the mechanism of Syk survival signaling in EBV+ B cell lymphomas from post-transplant lymphoproliferative disorder (PTLD) to discover virally controlled therapeutic targets involved in lymphomagenesis and tumor progression. Using small molecule inhibition and siRNA strategies, we show that Syk inhibition reduces proliferation and induces apoptosis of PTLD-derived EBV+ B cell lines. Syk inhibition also reduces autocrine IL-10 production. Although Syk inhibition attenuates signaling through both the PI3K/Akt and Erk pathways, only PI3K/Akt inhibition causes apoptosis of PTLD-derived cell lines. Loss of the endogenous caspase inhibitor XIAP is observed after Syk or PI3K/Akt inhibition. The loss of XIAP and apoptosis that results from Syk or PI3K/Akt inhibition is reversed by inhibition of the mitochondrial protease HtrA2. Thus, Syk drives EBV+ B cell lymphoma survival through PI3K/Akt activation, which prevents the HtrA2-dependent loss of XIAP. Syk, Akt, and XIAP antagonists may present potential new therapeutic strategies for PTLD through targeting of EBV-driven survival signals.